Studies by several groups, initiated more than 30 years ago, revealed that immune complexes infused into animals are either rapidly phagocytosed by macrophages in the spleen and/or liver, or taken up by resident, but not antigen specific, B cells in the spleen and then later transferred to follicular dendritic cells (FDC). (Humphrey and Frank, Immunology, 13, 87-100 (1967); Brown et al., Immunology, 24, 955-968 (1973); Papamichail et al., Scandanavian Journal of Immunolgy, 4, 343-347 (1975); Klaus, Immunology, 34, 643-652 (1978); Humphrey et al., European Journal of Immunology, 14, 859-864, 1984; Heinen et al., European Journal of Immunolgy, 16, 167-172 (1986); van der Berg et al., European Journal of Immunolgy 22, 957-962 (1992)).
One component involved in the clearance of the infused immune complexes is the complement receptor 1 (CR1), present on primate erythrocytes. Research on the properties of CR1 has revealed that under a variety of conditions immune complexes (IC) comprising an antibody and antigen can be bound to this receptor either via complement opsonization or by the use of cross-linked complexes comprising monoclonal antibodies (mAbs) specific for CR1 itself (see U.S. Pat. Nos. 5,879,679 and 5,487,890, the disclosures of which are incorporated herein). Moreover, both in vivo and in vitro experiments have demonstrated that immune complexes bound to erythrocytes in either fashion are effectively transferred to acceptor macrophages in a reaction mediated by Fc receptors (“the transfer reaction”). During the transfer reaction CR1 is proteolyzed off of the erythrocytes, and then the entire immune complex and CR1 is internalized and destroyed by the acceptor macrophage. The key steps in the reaction include binding of the Fc regions of the antibodies in the immune complexes to Fc receptors on the macrophage, and then cleavage of CR1 by proteases associated with the macrophage, thus allowing the released immune complexes to be internalized by the macrophage.
These properties of primate erythrocyte CR1 have allowed the development of an invention comprising cross-linked mAb complexes for targeting circulating pathogens in the bloodstream. In particular, the cross-linked mAb complexes contain a mAb specific for erythrocyte CR1 cross linked with a mAb specific for a circulating pathogen. Pathogens present in the bloodstream become bound to such complexes upon administration of the complex to a patient, and the complexes themselves become bound to erythrocytes resulting in facilitated clearance of the pathogen via acceptor macrophages in the liver and spleen. The pathogen is then taken up by these acceptor cells and phagocytosed and destroyed, but the erythrocytes remain intact and are returned to the circulation.
Primate erythrocyte CR1 is similar in structure to complement receptor 2 protein (CR2), present on primate and non-primate B lymphocytes (B-cell). Both CR1 and CR2 are type I membrane-associated glycoproteins that are constructed with multiple copies of the short consensus repeat (SCR) folding motif, which is found in many complement control proteins. Both receptors have three separate domains: a cytoplasmic region, a transmembrane portion, and the main part of the protein, the extracytoplasmic domain, composed almost exclusively of multiple copies of the SCR.
Erythrocyte CR1 and B cell CR2 are both reduced in diseases, and in accordance with the present invention this is believed to result from the loss of the respective proteins during immune complex processing due to the transfer reaction. The immune complexes when bound to erythrocytes or B cells contain antigens, multiple copies of IgG, and either C3b when they are bound to erythrocytes, or C3dg or C3bi when they are bound to B cells. The fact that the complexes contain IgG allows them to interact with Fc receptors on the acceptor macrophages. Although the mechanism for antigen transfer from B cells to follicular dendritic cells (FDC) was never elucidated, it requires an intact complement system which promotes C3dg opsonization of the immune complexes.
As described in the present application, applicants have discovered that substrates bound to human B cells are capable of undergoing a CR2 mediated transfer reaction. This observation led to the present application, directed to bispecific reagents comprising a mAb specific for CR2 linked either directly or indirectly to a target antigen. The target antigen can be selected from any antigenic non-immunological component of a compound, cell or organism that one desires to effectively raise an immune response against or eliminate from the body of a vertebrate species. The bispecific antibody complexes of the present invention can be used to either present a particular antigen to a host organism's immune system, and thus induce a prophylactic immune response, or the bispecific antibody complex can be used to treat a pathogenic infection.
The bispecific antibody complexes of the present invention and their use in the present invention follows the natural mechanism for handling of immune complexed antigens which occurs in the body of warm-blooded vertebrates. In the natural process, complement labels the immune complex with C3dg, the immune complexes are then bound to B cell CR2, and finally transferred to FDC or macrophages. In the present invention, the monoclonal antibody specific for CR2 serves as a surrogate for C3dg and therefore insures efficient binding of substrates to B cell CR2, as well as later transfer of the antigen to the FDC. Under these conditions complement is not even required to achieve localization of the antigen to the FDC. The natural pathway is mediated by a reaction of highly variable efficiency: C3dg opsonization of immune complexed antigens. The present invention removes this uncertainty in antigen targeting, as the anti-CR2 mAb works far more effectively.